How do I determine the titer of my viral stock?

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Answer

We recommend you perform a plaque assay to determine the titer of your viral stock. You may also perform a plaque assay to purify a single viral clone, if desired.

Answer Id: E9423

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Can I perform the single-step protocol for the BP/LR Clonase™ reaction using BP Clonase™ enzyme and LR Clonase™ enzyme instead of BP Clonase™ II enzyme and LR Clonase™ II enzyme?

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Answer

In the single-step protocol for the BP/LR Clonase™ reaction, we would not recommend substituting the BP Clonase™ II/LR Clonase™ II enzymes with BP Clonase™ /LR Clonase™ enzymes as this would result in very low recombination efficiency.

Answer Id: E9858

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What size targets can be amplified by the SuperScript™ One-Step RT-PCR Systems?

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Answer

The SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is recommended for amplifying RNA targets up to 4.5 kb and the SuperScript™ II One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is recommended for amplifying RNA targets up to 3.5 kb. Even though the SuperScript™ II and III One-Step RT-PCR Systems with Platinum™ Taq High Fidelity can amplify smaller targets, it is recommended for amplifying RNA targets from 1 kb up to 9 kb.

Answer Id: E3213

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I’m yielding no plaques from my baculovirus plaque assay. What are the possible causes for this?

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Answer

The kinetics of infection may be slower than expected. Observe plates until the 8-9th day after infection. If no plaques appear, investigate the following:

- If the cells are not healthy, then poor-quality or no plaques can result. Ideally, cells should be in mid-log phase and have a viability of greater than 90%. Cells should double at least once before infection stops growth. Ensure that the correct amount of cells was used at ~70% confluency.
- The viral replication cycle can be inhibited due to poor nutritional and physical conditions of the cell.
- The temperature of the agarose is also crucial. After overlaying the agarose, the plates should be left untouched for 1 hour for the agarose to completely solidify.
- Excessive condensation during incubation at 27 degrees C can inhibit plaque formation-remove paper towels or open the container containing plates as soon as condensation appears.
- The viral titer is too low: Use a higher viral titer. You may need to re-infect your cells and collect a higher titer of your viral stock.

Answer Id: E9465

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Can you outline the main steps of performing a plaque assay, and any suggestions when performing this assay?

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Please see the method below for an outline of the main steps of performing a plaque assay:

- Plate cells at 80% confluency in a 6-well plate
- Make a serial dilution of the P1 viral stock (1-10-5) and add to cells
- Incubate for an hour at 27 degrees C
- Mix 1% melted agarose into the medium
- Remove the viral supernatant
- Overlay the cells with the medium containing agarose
- Leave the plates for 2-3 hours for agar to completely solidify
- Incubate plates for 10-14 days
- Count plaques

When performing this assay, we suggest:

- Use cells that are in excellent health, of low passage (10-20) in log-phase growth, and high viability (>95%)
- Check viral stock for sterility (free of contamination)
- Use high-quality, low melting point agarose
- The temperature of the medium with agarose is crucial-too hot, cells will die; but if too cold, it will solidify too quickly
- Wait 2-4 hours before removing the plate after overlay so that the agarose can 100% solidify
- Count plaques on a dilution plate where (1/dilution) x # of plaques = pfu/mL
e.g., if you have 50 plaques on the 10-6 plate, then you have 1(10-6) x 50 = 5 x 10e7 pfu/mL

Answer Id: E9424

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How does adding Platinum™ Taq DNA Polymerase improve SuperScript™ One-Step RT-PCR performance?

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Answer

Platinum™ Taq DNA Polymerase is precomplexed with a mixture of antibodies that inhibit polymerase activity until the initial denaturation step in PCR. As a result, nonspecific polymerase acitivty at lower temperatures during set-up and reverse transcription is eliminated, which provides greater yield and specificity of intended product.

Answer Id: E3214

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I think I’ve added too much virus. How important is the MOI? What happens if too much virus is used?

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Answer

An MOI of 5-10 is typically used. If too much virus is added, unfortunately the cells die too soon and the protein expression level goes down.

Answer Id: E9466

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What is the equation to calculate viral titer? Do you have an example I can use?

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Answer

Please see the equation below:
pfu/mL = number of plaques (pfu)/dilution factor x mL of inocula
So, if you have a well with viral dilution of 10-8 containing 18 white plaques, the viral titer is calculated as followed:
X pfu/mL = 18 pfu/10-8 x 1 mL
X = 1.8 x 10e9 pfu/mL

Answer Id: E9425

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Can the attB primers anneal in a non-specific manner?

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Answer

No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.

Answer Id: E3199

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What is the smallest quantity of RNA detectable by the SuperScript™ First-Strand System for RT-PCR?

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Answer

Detection limits dependend on many factors, including primer design, target size, and the abundance of message. In our hands, this system was able to detect GAPDH mRNA from as little as 1.0 pg of total HeLa RNA when used in conjunction with Platinum™ Taq DNA Polymerase High Fidelity.

Answer Id: E3215

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I’d like to perform PCR on a plaque, and also make a stock from the same plaque. How do you suggest I do this?

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Answer

Our R&D team will typically pick a plug and add it to a 12-well dish with 0.5 x 10e6 cells/well and 2.5 mL total volume per well. After approximately 3 days, remove 0.75 mL to make DNA for PCR and keep the remaining medium in an Eppendorf tube as your P1 viral stock. As an aside, it is okay to pick a plaque and store it in Grace’s medium.

Answer Id: E9431

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Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

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Answer

We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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I see what looks like small plaques, but they are too difficult to visualize. What could be the problem?

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Answer

Too many cells were seeded; we recommend seeding 8 x 10e5 cells per well for a 6-well plate.

Answer Id: E9467

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What might cause plates to turn blue along with blue plaques?

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Answer

There are a few things that can turn plates blue:

- Too much virus when plating. Try a higher dilution.
- Cells are being singed when plated with hot melted agarose. This lyses the cells and releases lacZ into the agarose, turning it blue. Double-check plating temperatures. If plates are too wet, the blue can diffuse.

Answer Id: E9470

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I’ve infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

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Answer

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

Answer Id: E9473

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